Method for producing natively folded proteins in a prokaryotic host

ABSTRACT

The present invention relates to a method for producing a protein of interest containing one or more disulfide bonds in its native state. The method comprises that a prokaryotic host cell is genetically engineered to express the protein of interest and a sulfhydryl oxidase in the cytoplasm of the host cell. The protein of interest is formed in a soluble form and contains disulfide bonds due to the presence of the sulfhydryl oxidase in the cytoplasm of said host cell. The present invention relates also to a prokaryotic host cell and a vector system for producing a protein of interest containing natively folded disulfide bonds.

PRIORITY CLAIM

This is a continuation-in-part application of International applicationnumber PCT/FI2010/05448 filed on Jun. 2, 2010 claiming priority of theFinnish national patent application number 20095615 filed on Jun. 2,2009, the contents of both of which are incorporated herein by referencein their entirety.

SEQUENCE DATA

This application contains sequence data provided in computer readableform and as PDF-format. The PDF-version of the sequence data isidentical to the computer readable format.

FIELD OF THE INVENTION

This invention relates to a method, a host cell and a vector system forproducing a protein of interest containing one or more disulfide bondsin its native state. In particular, the invention relates to a method, ahost cell and a vector system for producing such proteins in aprokaryotic host.

DESCRIPTION OF RELATED ART

Many proteins and enzymes of biotechnological importance containstructure stabilizing disulfide bonds, with an estimated one third ofall human proteins folding in the endoplasmic reticulum (ER) andacquiring disulfide bonds there. This includes most proteins which getsecreted or end up on the outer membrane. Since any two cysteineresidues in a protein have the potential to form a disulfide bond, thecorrect formation of native disulfide bonds is not trivial. Hence, it isunsurprising that native disulfide bond formation is often therate-limiting step in the folding of proteins in vitro and in vivo.

The process of native disulfide bond formation in the endoplasmicreticulum (ER), periplasm or inter-membrane space of mitochondria isknown to be catalysed by several families of enzymes. However, whilesome of the participants in the cellular process are known, theirprecise individual roles are still largely confused.

What is known is that native disulphide bond formation can occur viamultiple parallel pathways (see FIG. 1). The current state of the artsays that the major pathway for disulfide bond formation in the ER isvia oxidation of protein disulfide isomerase (PDI) family members by thesulfhydryl oxidase activity of the gene product(s) of ERO1 familymembers. PDI in turn then introduces disulfide bonds into foldingproteins.

Parallel pathways, for example via glutathione, are also possible.Similarly in the inter-membrane space of mitochondria a similar pathwayexists via the oxidation of Mia40 by the sulfhydryl oxidase Erv1p. Mia40in turn introduces disulfide bonds into folding proteins. A similarpathway exists in the periplasm of prokaryotes via DsbA and DsbB exceptthat DsbB is not a sulfhydryl oxidase.

During disulfide bond formation many non-native disulfide bonds may beformed. Hence in vitro in a glutathione redox buffer, and probably invivo for many proteins, the rate-limiting step for native disulfide bondformation is late-stage isomerization reactions, where disulfide bondisomerization is linked to conformational changes in protein substrateswith substantial regular secondary structure. These steps are catalysedby thiol-disulfide isomerases, in particular in the ER by proteinsbelonging to the PDI-family and in the periplasm of prokaryotes by DsbCand DsbG.

Currently proteins that contain disulfide bonds are difficult for thebiotech industry to produce on a large scale. The most common route isto produce these proteins in the cytoplasm of E. coli. Here there are nomechanisms for disulfide bond formation. Due to this the recombinantproteins are unable to attain their native conformation and forminsoluble inclusion bodies. Inclusion body refolding is a widely studiedand widely patented field. However, it is costly, complex and generallyinefficient. Alternative routes for producing disulfide bonded proteinsalso have drawbacks.

-   -   I. Disulfide bond formation in the periplasm of E. coli. While        native E. coli disulfide bond containing proteins fold        efficiently in the periplasm, the yields of heterologously        expressed proteins are often very low, in part due to the small        size of the periplasm. In addition, the outer membrane of E.        coli is freely diffusible to most small molecules which mean        that the biophysical environment of the periplasm is very        dependent on the external media.    -   II. Disulfide bond formation in the cytoplasm of modified E.        coli. E. coli has two pathways to ensure that its cytoplasm is        reducing: i) using thioredoxins/thioredoxin reductases and ii)        using glutathione/glutaredoxin/glutathione reductase (see FIG.        2). When both pathways are knocked out, for example in the        commercial origami or rosetta-gami strains (Novagen®), the        cytoplasm is less reducing and disulfide bonds form in proteins.        However, disulfide bond formation is still slow and inefficient.        In addition, these strains are less genetically stable and grow        significantly more slowly than wild type strains. While some        disulfide bond containing proteins can be formed in the        cytoplasm of origami or rosetta-gami (and equivalent), the        yields of most proteins are below that required for commercial        production.    -   III. Disulfide bond formation in the ER of eukaryotic organisms        such as S. cerevisiae, Pichia pastoris, insect cell culture or        even mammalian cell culture is much more efficient than that in        bacteria. However, there is the corresponding increase in costs        associated with the growth of eukaryotic organisms and problems        associated with the large scale production of proteins in cell        culture.

WO 9907727 A1 (or U.S. Pat. No. 6,361,964) describes a method ofincreasing disulfide bond formation in a protein by expressing theprotein in a host cell that also expresses an isolated nucleic acid thatencodes an Ero1 polypeptide or optionally an Ero1 polypeptide togetherwith a protein disulfide isomerase. The Ero1 polypeptide is suggested tobe for use in eukaryotic expression systems, wherein Ero1 aids foldingin the ER, or in vitro refolding reactions.

WO 2005061718 A1 describes a method for producing a heterologous proteinin a host cell utilizing a 2 micron-family plasmid. The host cell iscultured in such conditions that allow the expression of the geneencoding the fungal molecular chaperone or protein folding catalyst,such as Ero1 or protein disulfide isomerase, and the gene encoding aheterologous protein. The method uses 2 micron-family plasmids which canbe applied only to eukaryotic cells. Both the protein and the molecularchaperone or protein folding catalyst such as Ero1 are targeted to theER.

WO 2009058956 A1 describes a method for expressing a protein in afilamentous fungal host, wherein Ero1 can be co-expressed with thedesired protein. Ero1 facilitates the folding of the desired protein inthe ER.

KR 20070041166 A describes a method for producing a protein in the ER ofa yeast cell, wherein Ero1 and protein disulfide isomerase aresimultaneously expressed in a host cell.

Furthermore, U.S. Patent Application No. 2007/0193977 describes a methodfor producing a desired protein in a host cell comprising recombinantgenes for a first and a second chaperone and for the desired protein. Apreferred chaperone is PDI, in particular from fungal or mammalianorigin. According to the publication various host expression systemsincluding yeast, bacteria and mammalian cells may be used. Bacterialhost cells are mentioned to be useful for cloning purposes.

Examples of commercial proteins that are produced by these variousroutes include insulin, tissue plasminogen activator, growth hormonesand single chain antibodies. None of the mentioned patent publicationsdisclose a prokaryotic expression system for the production of nativelyfolded disulfide bond containing proteins.

SUMMARY

It is an object of the invention to provide a method for producing anatively folded disulfide bond containing protein in a prokaryotic host.

It is also an object of the invention to provide a host cell forproducing a natively folded disulfide bond containing protein in aprokaryotic host.

Further, it is also an object of the present invention to provide avector system for producing a natively folded disulfide bond containingprotein in a prokaryotic host. These and other objects together with theadvantages thereof over known methods, hosts and vectors, are achievedby the present invention as hereinafter described and claimed.

The invention is based on the use of genetic engineering of prokaryotichost cells to cytoplasmically express a sulfhydryl oxidase.

In one aspect, the present invention provides a method for producing aprotein of interest containing one or more disulfide bonds in its nativestate in a prokaryotic host.

In one embodiment the invention provides a method which comprises that aprokaryotic host cell is genetically engineered to express the proteinof interest and a sulfhydryl oxidase in the cytoplasm of the host cell,said protein of interest being formed in a soluble form and containingdisulfide bonds due to the presence of the sulfhydryl oxidase in thecytoplasm of said host cell.

In one embodiment of the invention the protein product comprising theprotein of interest is recovered from the cell culture or from the hostcells and optionally purified. In another aspect, the present inventionprovides a prokaryotic host cell for producing a protein of interestcontaining one or more disulfide bonds in its native state in aprokaryotic host.

In one embodiment the invention provides a prokaryotic host cell forproducing a protein of interest containing natively folded disulfidebonds, which comprises that the host cell is genetically engineered toexpress a sulfhydryl oxidase and a protein of interest in the cytoplasmof the host cell.

In one preferred embodiment of the invention the sulfhydryl oxidase isco-expressed with the protein of interest.

In another preferred embodiment the sulfhydryl oxidase is expressedprior to the protein of interest.

In one further preferred embodiment, the prokaryotic host cells areengineered to express also a thiol-disulfide isomerase in the cytoplasm.

In one still further preferred embodiment the thiol-disulfide isomeraseis the eukaryotic ER-resident enzyme protein disulfide isomerase (PDI).

In one further preferred embodiment the thiol-disulfide isomerase isDsbC, which is usually targeted to the periplasm of prokaryotes.

These co-expressions may be used separately or in combination with anyof the other variations described herein, so long as a sulfhydryloxidase is expressed in the host cell.

The host can be any prokaryotic host. In one embodiment of theinvention, the prokaryotic host is a bacterial host, in one specificembodiment a gram-negative host, such as E. coli.

In one further preferred embodiment, expression of the disulfide bondcontaining protein of interest is achieved by expressing the protein asa genetic fusion with a fusion partner. This may be used separately orin combination with any of the other variations described herein, solong as a sulfhydryl oxidase is expressed in the host cell.

In one further embodiment the host cell may be deficient in thioredoxinreductase or glutathione reductase activity. This may be caused byhaving a mutation or deletion in trxB gene and/or a mutation or deletionin gor gene. These deficiencies may be used separately or in combinationwith any of the other variations described herein, so long as asulfhydryl oxidase is expressed in the host cell.

In still further embodiments, codon usage for the nucleotide sequenceencoding the protein may be optimized for expression in a particularhost or the host codon usage manipulated so as to optimize expression ofthe recombinantly expressed protein. This may be done for example bysupplementing the expression host's tRNA levels by production of tRNAspecies encoded by engineered plasmids. Codon usage may also beoptimized for the sulfhydryl oxidase and/or the thiol-disulfideisomerase. These modifications may be used separately or in combinationwith any of the other variations described herein, so long as asulfhydryl oxidase is expressed in the host cell.

The present invention provides also a vector system, comprising one ormore vectors encoding sulfhydryl oxidase and the protein of interest,and optionally also, a thiol-disulfide isomerase. The proteins may beseparately inducible.

Hence the invention has possible alternative solutions, all with thecommon factor of cytoplasmic expression of a sulfhydryl oxidase.

The method of the present invention results in the formation of nativedisulfide bonds in a significant proportion. The method is particularlysuitable for expressing proteins where a biological activity isdependent on the formation of one or more intra- or inter-chaindisulfide bonds.

In the following, the invention will be examined more closely with theaid of a detailed description and with reference to some workingexamples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows pathways for disulfide bond formation in: Panel A: Theendoplasmic reticulum; Panel B: The inter-membrane space of mitochondria(protein names given for the system in Saccharomyces cerevisiae); PanelC: The periplasm of prokaryotes (protein names given for the system inEscherichia coli).

FIG. 2 shows the pathways for reducing disulfide bonds in the cytoplasmof E. coli. Gene names are given in parentheses. In commercial strainsfor the production of disulfide bonded proteins, such as origami androsetta-gami (Novagen®), both the thioredoxin and glutathione pathwaysare knocked out by gor and trxB deletions or knock-outs by transposonmutagenesis.

FIG. 3 shows protein sequences of representative members of the ERO1family of sulfhydryl oxidases (EC 1.8.4.-). The cleavable signalsequences are underlined. Endoplasmic oxiodreductin-1 from Saccharomycescerevisiae corresponds to SEQ ID NO: 1; Human ERO1-like protein alphacorresponds to SEQ ID NO:21 and Human ERO1-like protein beta correspondsto SEQ ID NO:3.

FIGS. 4A and 4B show protein sequences of representative members of theERV/ALR sulfhydryl oxidase domain containing sulfhydryl oxidases (Enzymeclassification: EC 1.8.3.2). The ERV/ALR domain sequences for each, ascurrently defined in UniProt, are underlined. FIG. 4B shows also proteinsequences of thiol-disulfide isomerases (EC 5.3.4.1) PDI and DsbC. Thesignal sequences are underlined. In FIG. 4 A, Human ALR corresponds toSEQ ID NO:4, S. cerevisiae mitochondrial FAD-like sulfhydryl oxidaseERV1 corresponds to SEQ ID NO:5, S. cerevisiae FAD like sulfhydryloxidase ERV2 corresponds to SEQ ID NO 6, and Human sulfhydryl oxidase 1corresponds to SEQ ID NO:7. In FIG. 4B Human sulfhydryl oxidsase 2corresponds to SEQ ID NO:8, Vaccinia virus FAD-linked sulhydryl oxidesE10 corresponds to SEQ ID NO 5, E. coli DsbC corresponds to SEQ ID NO:10and Human PDI corresponds to SEQ ID NO:11.

FIG. 5 shows plasmids pET23 and pLysS as modified for producing nativelyfolded disulfide bond containing proteins in these studies. The order ofthe genes on polycistronic vectors does not affect the ability toco-expression of the proteins.

FIG. 6 shows representative SDS-PAGE from expression of the luminaldomain of human tissue factor with co-expression with a sulfhydryloxidase in LB media at 25° C. Lane 2 shows the total E. coli lysate,lane 1 shows the soluble fraction. The position of the luminal domain oftissue factor is marked with an arrow.

FIG. 7 shows production of PhoA in the cytoplasm of E. coli in LB mediaat 30° C. Panel A) SDS-PAGE analysis. Alternate lanes show total (T) andsoluble (S) fractions. The final lanes show the purified protein fromBL21 with co-expression of Erv1p and the molecular weight markers. Thestrains used were BL=BL21 (DE3) pLysRARE and RG=rosetta-gami;+E=co-expression of the sulfhydryl oxidase Erv1p from a polycistronicvector. The positions of PhoA and Erv1p are marked with arrows. PanelB). Measurements for the activity of E. coli alkaline phosphataseexpressed in the cytoplasm of E. coli in LB media at 30° C.4-nitrophenylphosphate used as the substrate, pH of the reaction was 8.0and the values are cited as relative activity (%) compared with the mostactive system which is BL21 (DE3) pLysSRARE+expression of the sulfhydryloxidase. The strains used were BL=BL21 (DE3) pLysRARE andRG=rosetta-gami; +E=co-expression of the sulfhydryl oxidase Erv1p from apolycistronic vector. Panel C) Representative blot from a shift-assaybased on alkylation of free thiol groups to examine the disulfide bondstatus of the PhoA produced. The samples are treated with thethiol-blocking agent N-ethylmaleimide (NEM) before reduction andmaleimide based addition of polyethyleneglycol. Hence an increase inapparent molecular weight is consistent with the presence of one or moredisulfide bonds in the original sample. Panel D) Specific activity(μmole of product formed per minute per mg of protein) of PhoA purifiedfrom E. coli strains with and without co-expression of a sulfhydryloxidase. The data is shown as mean±s.d.

FIG. 8 shows production of AppA in the cytoplasm of E. coli strains inLB media at 30° C. Panel A) Measurements for the activity of E. coliphytase (AppA). Absorbance values were measured after quenching thereaction with sodium hydroxide (NaOH) and the values are cites asrelative activity (%) compared with the most active system which is BL21(DE3) pLysSRARE+expression of the sulfhydryl oxidase and DsbC. Thebackground activity is subtracted. Strains used were BL=BL21 (DE3)pLysSRARE and RG=rosetta-gami. +=co-expression from a polycistronicvector where D=mature E. coli DsbC, E=S. cerevisiae Erv1p. Panel B)Representative blot from a shift-assay based on alkylation of free thiolgroups to examine the disulfide bond status of the AppA produced.

FIG. 9 shows SDS-PAGE from expression of the human CSF3 (lanes 2-7) andBMP4 (lanes 8-13) as fusion proteins with MBP in the cytoplasm of E.coli with co-expression with a sulfhydryl oxidase in LB media at 30° C.and co-expression of PDI (lanes 4,5,10,11) or DsbC (lanes 6,7,12,13).Even numbered lanes show the total E. coli lysates, odd numbered lanesshow the soluble fractions (except lane 1 which shows molecular weightmarkers). The position of the MBP fusion proteins CSF3 and BMP4 aremarked with arrows.

FIG. 10 shows analysis of human kringle tPA expressed in various systemsin LB media at 30° C. Panel A) shows measurements for the activity ofhuman kringle tPA with either an N-terminal His-tag or N-terminalMBP-tag expression in the cytoplasm of E. coli. BL21 pLysS RARE (BL) orrosetta-gami (RG) or origami (OG), with co-expression with thesulfhydryl oxidase Erv1p and/or DsbC. The data is normalized to theequivalent of the best previously reported system (a ΔtrxB Δgor strainwith co-expression of mature DsbC) Panel B) SDS-PAGE from expression ofthe human kringle tPA as a fusion protein with MBP in the cytoplasm ofE. coli. BL21 pLysS RARE (BL) or rosetta-gami (RG) or origami (OG), withco-expression with the sulfhydryl oxidase Erv1p and DsbC. Alternatelanes show the total E. coli lysates (T), and the soluble fractions (S).The position of the MBP-kringle tPA fusion protein is marked with anarrow. Panels C-E) show the results from measurements for the activityof the MBP-kringle t-PA fusion protein.

FIG. 11 shows analysis of BPTI production in origami+pre-expression ofthe sulfhydryl oxidase and PDI in LB media at 30° C. Panel A) SDS-PAGEfrom expression of BPTI wild type (lanes 1 and 2), along with theC49A/C73A (lanes 3 and 4) and C65A/C86A (lanes 5 and 6) mutants, whichshould contain only 2 disulfide bonds, in the cytoplasm of origami E.coli with pre-expression of the sulfhydryl oxidase (SO) and PDI. Oddnumbered lanes show the total E. coli lysates, even numbered lanes showthe soluble fractions. The position of BPTI is marked with an arrow.Panel B) Purification of the C49A/C73A or C65A/C86A mutants of BPTIusing immobilized metal affinity chromatography and subsequent analysisby reducing (lanes 1 and 3) and non-reducing (lanes 2 and 4) SDS-PAGEindicated that only monomeric species were obtained for the C49A/C73A(lanes 1 and 2) and for the C65A/C86A (lanes 3 and 4) BPTI mutants i.e.no mixed disulfides were formed. Panel C) Analysis of the C49A/C73Amutants using immobilized metal affinity chromatography and subsequentanalysis by reverse phase HPLC analysis. The elution positions of thenative species containing 3 disulfides, the fully reduced species andthe intermediate species containing only 1 or 2 disulfides areindicated. These results indicate that only a single two disulfidespecies is obtained.

FIG. 12 shows analysis of the production of human interferon α2 andinterleukin 17 in E. coli with co- or pre-expression of a sulfhydryloxidase.

FIG. 13 shows analysis of resistin expressed in the cytoplasm of E. coliwith or without the pre-expression of a sulfhydryl oxidase and DsbC.Panel A) SDS-PAGE analysis of purified MBP-resistin under reducing andnon-reducing conditions. Panel B) SDS-PAGE analysis of mature resistinproduced in origami with pre-expression of a sulfhydryl oxidase andDsbC.

FIG. 14 shows reverse phase HPLC analysis of ¹⁵N labeled interleukin 6and human growth hormone 1 produced in ¹⁵N-labelled M9 minimal mediawith co-expression of a sulfhydryl oxidase.

SEQUENCE LISTING

The sequence listing includes the sequences:

SEQ ID NO: 1 Endoplasmic oxidoreductin-1 from Saccharomyces cerevisiae(Swiss-Prot ID Q03103)

SEQ ID NO: 2 Human ERO1-like protein alpha (Swiss-Prot ID Q96HE7)

SEQ ID NO: 3 Human ERO1-like protein beta (Swiss-Prot ID Q86YB8)

SEQ ID NO: 4 Human ALR (Swiss-Prot ID P55789)

SEQ ID NO: 5 S. cerevisiae mitochondrial FAD-linked sulfhydryl oxidaseERV1 (Swiss-Prot ID P27882)

SEQ ID NO:6 S. cerevisiae FAD-linked sulfhydryl oxidase ERV2 (Swiss-ProtID Q12284)

SEQ ID NO: 7 Human sulfhydryl oxidase 1 (QSOX; Swiss-Prot ID 000391)

SEQ ID NO: 8 Human sulfhydryl oxidase 2 (Swiss-Prot ID Q6ZRP7)

SEQ ID NO: 9 Vaccinia virus FAD-linked sulfhydryl oxidase E10(Swiss-Prot ID P21050)

SEQ ID NO: 10 E. coli (K12 strain) DsbC

SEQ ID NO: 11 Human PDI

SEQ ID NO: 12 hexa-histidine tag MHHHHHHM

SEQ ID NO: 13 linker sequence NSSSNNNNHM

SEQ ID NO: 14 linker sequence GSGSGSGSGSIEGRGSGSGSGSGSHM

DETAILED DESCRIPTION OF THE INVENTION Definitions

“GSH” means reduced glutathione, a tripeptide called alsoγ-glutamylcysteinylglycine.

“GSSG” means oxidized glutathione. In GSSG two GSH's are linked by adisulfide bond.

“Oxidases” mean here enzymes that can use molecular oxygen to oxidize a(bio)chemical compound.

“Thioredoxins” mean enzymes that catalyse the reduction of disulfidebonds, which are often found in protein substrates. Thioredoxin isreduced in turn by thioredoxin reductase.

“Glutaredoxins” mean enzymes that catalyse the removal of glutathionefrom glutathione-protein mixed disulfides using reduced glutathione andgenerating oxidized glutathione, which in turn is reduced by glutathionereductase.

“PDI” mean protein disulfide isomerase. PDI's are enzymes which catalysethiol-disulfide exchange reactions in the endoplasmic reticulum (ER) ofeukaryotes. They belong to the thioredoxin superfamily.

“Dsb” mean disulfide bond forming enzymes. Dsb's are enzymes whichcatalyse thiol-disulfide exchange reactions in the periplasm ofprokaryotes. DsbA, DsbC and DsbG belong to the thioredoxin superfamily.DsbB and DsbD are not thiol-disulfide isomerases.

Thioredoxin superfamily members are for example thioredoxins, proteindisulfide isomerases (PDI's) and disulfide bond forming enzymes (Dsb's).

The present invention relates to a method for producing a nativelyfolded disulfide bond containing protein in a prokaryotic host. Themethod comprises that a prokaryotic host cell is genetically engineeredto express the protein of interest and a sulfhydryl oxidase in thecytoplasm of the host cell. The protein of interest is formed in asoluble form and contains disulfide bonds due to the presence of thesulfhydryl oxidase in the cytoplasm of said host cell. The presentinvention relates also to a prokaryotic host cell and a vector systemfor producing a protein of interest containing natively folded disulfidebonds.

A common prejudice in the field has been that the cytoplasm of mostprokaryotes is reducing and therefore one cannot make disulfide bondcontaining proteins in the cytoplasm of a prokaryotic host unless thepathways for the reduction of disulfide bonds are disrupted or deleted,such as a ΔtrxB/Δgor strain. No one has disclosed introducing an activecatalyst of de novo disulfide bond formation into the cytoplasm of aprokaryotic host.

Pathways for disulfide bond formation in the endoplasmic reticulum,inter-membrane space of mitochondria and periplasm of prokaryotes areshown in FIG. 1. Multiple pathways for dithiol oxidation to a disulfidein a folding protein exist. Direct oxidation by molecular oxygen, whilewidely used in vitro, is too slow to have physiological significance innative disulfide bond formation in vivo.

In the ER, PDI family members and GSSG can both introduce disulfidebonds into folding proteins, but both need to be reoxidised to completethe catalytic cycle. Oxidation of both PDI-family members andglutathione by molecular oxygen is too slow to have physiologicalsignificance in native disulfide bond formation in vivo. PDI is thoughtto be reoxidised by the sulfhydryl oxidase activity of Ero1-familymembers while the source of GSSG is under debate in the literature. Thehydrogen peroxide made by Ero1 each catalytic cycle has the potential tooxidise dithiols in substrate proteins, to oxidise reduced glutathioneto GSSG, to oxidise the active site of PDI family members and to formthe regulatory disulfides in Ero1 and hence shut down peroxideproduction.

In the inter-membrane space of mitochondria of Saccharomyces cerevisiaeMia40 introduces disulfide bonds into folding proteins. To complete thecatalytic cycle Mia40 is reoxidised by the sulfhydryl oxidase Erv1p. Thedirect formation of disulfide bonds in folding proteins or in Mia40 bymolecular oxygen is too slow to have physiological significance innative disulfide bond formation in vivo. The potential parallel role ofglutathione in disulfide bond formation in the inter-membrane space ofmitochondria is unknown.

In the periplasm DsbA introduces disulfide bonds into folding proteins.To complete the catalytic cycle DsbA is reoxidised by the transmembraneprotein DsbB. Unlike Ero1-family members or Erv1p, DsbB does not havesulfhydryl oxidase activity. The direct formation of disulfide bonds infolding proteins or in DsbB by molecular oxygen is too slow to havephysiological significance in native disulfide bond formation in vivo.The potential parallel role of glutathione in disulfide bond formationin the periplasm is under debate in the literature.

Herein, by “protein of interest” are meant in particular proteinsproduced recombinantly in a foreign host. The proteins contain one ormore disulfide bonds in their native state which are required to attaintheir native conformation. Many such proteins when expressed in a systemin which disulfide bond formation is limited form insoluble inclusionbodies within the host. A protein of interest is here typically aeukaryotic protein, usually a mammalian protein, in particular a humanprotein.

The protein may have in its native state multiple disulfide bonds. Themethod of the present invention is particularly suitable for producing aprotein having in its native state two or more disulfide bonds.

By “a host” or “a foreign host” is meant here in particular aprokaryotic host. More specifically the host can be a bacterial host, inparticular a gram negative host, such as Escherichia coli (E. coli). Inone preferred embodiment the host is E. coli.

According to this disclosure it is possible to produce a natively foldeddisulfide bond containing protein in a prokaryotic host. According to apreferred embodiment of the invention the protein may be recovered andoptionally purified from the cultured host cells. The protein may belyophilized or formulated with a carrier or diluents, if needed.

The advantage of the present invention is that the protein of interestis formed in soluble form. Typically no denaturation and renaturationsteps of the protein are needed. Furthermore, the protein is produceddirectly in biologically active form.

Commercially significant proteins which may be produced by using thepresent invention comprise for example insulin, blood coagulationfactors, cytokines, chemokines, interferons, growth hormones and singlechain antibodies.

In this disclosure as examples of such proteins are the luminal domainof human tissue factor, E. coli alkaline phosphatase and phytase, bovinepancreatic trypsin inhibitor (BPTI), human colony stimulating factor 3(CSF3), bone morphogenic protein 4 (BMP4), tissue plasminogen activator(t-PA), interferon α2, interleukin 6, interleukin 17, resistin andgrowth hormone 1.

In this disclosure the ability to generate “soluble protein” or“insoluble inclusion bodies” is deduced from SDS-PAGE analysis of totaland soluble fractions of a cell lysate. The generation of insolubleinclusion bodies is a common occurrence when proteins that nativelycontain disulfide bonds are expressed in the cytoplasm of prokaryotichosts. The formation of disulfide bonds allows protein folding to occurand hence allows the formation of soluble protein.

In this disclosure the “number of disulfide bonds” is deduced from thetotal number of cysteines in the protein and the number of cysteinesfree to react with iodoacetamide, as determined from mass spectrometricanalysis after treatment of the protein with iodoacetamide. The reactionof a thiol group with iodoacetamide adds 57 Da to the mass of theprotein.

In this disclosure the “biological activity” of a protein is deduced bywell known methods in the art appropriate for the individual proteinsbeing assayed. The biological activity or function of a protein reflectscharacteristics of the protein that result from the structure andconformational flexibility of the protein. These in turn are oftendependent on the formation of native disulfide bonds. Hence biologicalactivity, for example the ability of an enzyme to catalyze a specificenzymatic activity, is a measure of the attainment of the formation ofnative disulfide bonds within a protein.

The present invention provides a method for the production of proteinsthat require disulfide bond formation to reach their native biologicallyactive conformation. The methods presented are particularly suitable forthe expression of biologically active proteins that require theformation of multiple disulfide bonds. By multiple disulfide bonds ishere meant two or more than two, typically more than three disulfidebonds.

By “a sequential disulfide bond” is meant a covalent linkage between thesulfur atoms of two cysteine residues in a protein which do not have anintervening cysteine residue in the primary sequence of the proteinwhich is involved in another disulfide bond.

By “a non-sequential disulfide bond” is meant a covalent linkage betweenthe sulfur atoms of two cysteine residues in a protein which have anintervening cysteine residue in the primary sequence of the proteinwhich is involved in another disulfide bond.

The present invention is based on the use of pre- or co-expression of asulfhydryl oxidase in the cytoplasm of prokaryotic bacteria, preferablyof gram-negative bacteria such as E. coli, to generate disulfide bondsin folding proteins. As used herein, “sulfhydryl oxidases” refer toenzymes which use molecular oxygen to catalyze the reaction:

dithiol + O₂ → disulfide + H₂O₂

Since hydrogen peroxide (H₂O₂) is an oxidant that can be used to makedisulfide bonds the overall reaction can also be written:

4  thiols + O₂ → 2  disulfides + 2  H₂O

In addition, some sulfhydryl oxidases, such as Erv1, are reported alsoto be able to transfer electrons to the cytochrome respiratory chain inmitochondria instead of using molecular oxygen as the acceptor.

Sulfhydryl oxidases comprise proteins such as Ero1, Erv1, Erv2, QSox,ALR etc. These enzymes use FAD as a cofactor and contain one, or more,redox active disulfide bonds. They fall into two families:

-   -   i) the Ero family (Enzyme classification: EC 1.8.4.-) which are        endoplasmic reticulum located sulfhydryl oxidases and which        comprises, but is not limited to, the following exemplary        members (see FIG. 3 for sequences): Endoplasmic oxidoreductin-1        from Saccharomyces cerevisiae (Swiss-Prot ID Q03103; for example        as Genbank accession number CAA90553) (SEQ ID NO:1), human        ERO1-like protein alpha (Swiss-Prot ID Q96HE7; for example as        Genbank accession numbers AAF35260 or AAQ88828 or BAF85528 or        AAH08674 or AAH12941) (SEQ ID NO:2), and human ERO1-like protein        beta (Swiss-Prot ID Q86YB8; for example as Genbank accession        numbers AAF97547 or CAI23525 or CAI14420 or AAH32823 or        AAH44573) (SEQ ID NO:3);    -   ii) proteins which contain an ERV/ALR sulfhydryl oxidase domain        (Enzyme classification: EC 1.8.3.2). These proteins are found in        a variety of cellular compartments including mitochondria and        the secretory pathway. They include, but are not limited to, the        following exemplary members (see FIG. 4 for sequences): Human        ALR (Swiss-Prot ID P55789; for example as Genbank accession        numbers AAG38105) (SEQ ID NO:4), Saccharomyces cerevisiae        mitochondrial FAD-linked sulfhydryl oxidase ERV1 (Swiss-Prot ID        P27882; for example as Genbank accession numbers CAA97017) (SEQ        ID NO:5), Saccharomyces cerevisiae FAD-linked sulfhydryl oxidase        ERV2 (Swiss-Prot ID Q12284; for example as Genbank accession        numbers CAA94987 or CAA92143) (SEQ ID NO:6), Human sulfhydryl        oxidase 1 (QSOX; Swiss-Prot ID 000391; for example as Genbank        accession numbers AAC09010 or AAQ89300 or CAI14838 or AAI00024)        (SEQ ID NO:7), Human sulfhydryl oxidase 2 (Swiss-Prot ID Q6ZRP7;        for example as Genbank accession numbers CAI16881 or CAM28352)        (SEQ ID NO:8) and Vaccinia virus FAD-linked sulfhydryl oxidase        E10 (Swiss-Prot ID P21050; for example as Genbank accession        number AAA48051) (SEQ ID NO:9).

Within the scope of the present invention are proteins comprising any ofthe amino acid sequences of the above listed Genbank accession numbersor any of the amino acid sequences SEQ ID NO: 1 to 9, or a fragment ofsaid sequences or a modified version of said sequences, which sequencesstill have sulfhydryl oxidase activity.

“A modified protein” refers to a protein wherein at one or morepositions there have been amino acid insertions, deletions, orsubstitutions, either conservative or nonconservative, provided thatsuch changes result in a protein still having sulfhydryl oxidaseactivity.

By “conservative substitutions” are meant here combinations, such asVal, Ile, Leu, Ala, Met; Asp, Glu; Asn, Gln; Ser, Thr, Gly, Ala; Lys,Arg, H is; and Phe, Tyr, Trp. Preferred conservative substitutionscomprise Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr, Lys,Arg; and Phe, Tyr.

A modified version (or variant) of the sulfhydryl oxidase comprisestypically an amino acid sequence having at least 25%, preferably atleast 40%, more preferably at least 50%, still more preferably at least60%, still and still more preferably at least 70%, more and morepreferably at least 90% identity, most preferably at least 95%, or atleast 98% identity to any of the amino acid sequences of the abovelisted Genbank accession numbers or to any of the amino acid sequencesSEQ ID NO: 1 to 9.

In addition to full length sulfhydryl oxidases, shorter fragments may beused as long as they retain sulfhydryl oxidase activity. A fragmentrefers to a protein having at one or more positions deletions. Thefragment may comprise at least 30%, at least 40%, at least 50%,preferably at least 60%, more preferably at least 70%, still morepreferably at least 80%, more and more preferably at least 90%, or atleast 95% of the complete amino acid sequence of the proteins.

By the term “identity” is here meant the identity between two amino acidsequences compared to each other from the first amino acid encoded bythe corresponding gene to the last amino acid. Preferably the identityis measured by comparing the amino acid sequences without the sequencesof any signal peptide the protein may have. The identity of thefull-length sequences may be measured for example by using sequenceanalysis software, for example BLAST software available from theNational Library of Medicine.

According to other preferred embodiments of the invention, thesulfhydryl oxidase of the invention is encoded by a nucleic acidsequence hybridizing under low or under high stringent conditions tonucleic acid sequences encoding amino acid sequences of the above listedGenbank accession numbers or amino acid sequences SEQ ID NO: 1 to 9. Byhigh stringency conditions are meant conditions as disclosed for examplein Ausubel et al. Current Protocols in Molecular Biology, 1996, WileySons, New York, N.Y. High stringency hybridization conditions maycomprise hybridization at about 42° C. and about 50% formamide, a firstwash at about 65° C., about 2×SSC, and 1% SDS, followed by second washat about 65° C., and about 0.1% SDS, 1×SSC. Lower stringencyhybridization conditions may comprise hybridization at about 42° C. inthe absence of formamide, a first wash at about 42° C., about 6×SSC, and1% SDS, followed by second wash at about 50° C., about 6×SSC and about1% SDS. Within the scope of the present invention are also nucleic acidsequences encoding the proteins comprising any of the amino acidsequences of the above listed Genbank accession numbers or any of theamino acid sequences SEQ ID NO: 1 to 9, or nucleic acid sequencesencoding a fragment of said sequences or a modified version of saidsequences, which sequences still have sulfhydryl oxidase activity.Suitable nucleic acid sequences encoding proteins having sulfhydryloxidase activity are publicly available and can be found in gene banks.

Within the scope of the present invention are any enzymes withsulfhydryl oxidase activity. In particular, within the scope of thepresent invention are sulfhydryl oxidases comprising ERV/ALR sulfhydryloxidase domain, in particular Erv1.

Within the scope of the invention are sulfhydryl oxidases belonging toEro1 family proteins, in particular Ero 1, which is an ER-residentprotein having an N-terminal signal sequence, the signal sequence shouldbe removed before cytoplasmic expression is possible. Ero1 has multipledisulfide bonds which are required for it to function. It cannot fold toan active state in the cytoplasm of wild-type prokaryotes. Therefore,modifications, such as making the host cell deficient in thioredoxinreductase and/or glutathione reductase activity are needed to obtainfunctional protein, or preferentially the pre-expression of anothersulfhydryl oxidase such as Erv1p will generate functional Ero1 in thecytoplasm. Ero1 works in the endoplasmic reticulum of eukaryotes, acompartment which has a significantly more oxidizing redox state thanthe cytoplasm. This arises due to the presence of oxidized glutathione.In the cytoplasm any oxidized glutathione made (by any route) is reducedby glutathione reductase (the gor gene product).

The present invention has been exemplified here by using theSaccharomyces cerevisiae enzyme Erv1p. Erv1p is a 189 amino acidFAD-dependent sulfhydryl oxidase that catalyzes disulfide bondformation. It is required for the import and folding of smallcysteine-containing proteins in the mitochondrial inter-membrane spaceand is thought to form a redox cycle with Mia40.

In addition to wild type sulfhydryl oxidases, mutants may be used aslong as they retain sulfhydryl oxidase activity.

In addition to wild type sulfhydryl oxidases, chimeric fusion proteinsmay be used as long as they retain sulfhydryl oxidase activity.

Sulfhydryl oxidase according to the present disclosure is produced insoluble and biologically active form in the cytoplasm of the prokaryotichost.

Furthermore, sulfhydryl oxidase according to the present disclosure istypically capable of functioning without a partner protein e.g. Erv1pcan function without Mia40.

The present invention provides also a vector system comprising

-   -   a vector encoding a protein of interest, or having an insertion        site for a nucleic acid sequence encoding a protein of interest,        and a sulfhydryl oxidase, or    -   a first vector encoding a protein of interest, or having an        insertion site for a nucleic acid sequence encoding a protein of        interest, and    -   a second vector encoding a sulfhydryl oxidase.

According to the present disclosure the vector or vectors areconstructed to be capable of expressing the protein of interest and thesulfhydryl oxidase in the cytoplasm of the prokaryotic host cell.

The system may further comprise a vector encoding a thiol-disulfideisomerase in the cytoplasm of the prokaryotic host cell.

The vector encoding a thiol-disulfide isomerase may be the same vectorencoding the sulfhydryl oxidase and/or the protein of interest.

The vector system can be introduced to a suitable prokaryotic host celland the host can be cultured to produce a protein of interest comprisingnatively folded disulfide bonds.

The vector or vectors comprise a nucleic acid sequence (a gene) encodinga protein of interest and a sulfhydryl oxidase, and possible athiol-disulfide isomerase. Two or all of said nucleic acid sequences maybe in the same vector or all of them may be in different vectors. Thenucleic acid sequence may be linked to a nucleic acid sequence encodinga suitable part of a fusion protein, said nucleic acid constructionencoding a fusion protein. The vector system furthermore comprisesregulatory elements for multiplying and expressing the nucleic acidsequences in a prokaryotic host. Each mRNA produced from the vector(s)may be separately inducible. The vector system may comprise alsoselection markers.

Host cells comprising the expression vector encoding the sulfhydryloxidase and a protein of interest are cultured to produce the protein ofinterest in a biologically active form. Any suitable expression vectormay be used. It is preferable that the vector contains an appropriateselection marker. It is also preferable that the vector contains asystem for inducing expression.

Methods for cloning the genes of interest into appropriate vectors andculturing prokaryotic organisms are well known in the art.

The construction of suitable vectors has been exemplified here in theexamples which report the use of pET23 and pLysS plasmid derivatives.These have ampicillin and chloramphenicol selection markers,respectively. T7 and arabinose inducible expression systems were used.

Any suitable culture media may be used for the cultivation of theprokaryotic organisms. In the examples reported here Luria-Bertani Media(LB media) and M9 minimal media was used. Enbase media (Biosilta Oy) andautoinduction media has also successfully been used.

The protein may be obtained from the cultured cells in a soluble form byroutine cell lysis methods.

Cell lysis has been exemplified here in the examples by performing theaddition of 0.1 mg/ml lysozyme to the resuspended cell pellet followedby freeze-thawing.

The protein of interest can be isolated from the cell lysate insubstantially pure form by methods well known in the art and that areappropriate for the individual proteins and final application, forexample column chromatography, polyacrylamide electrophoresis, or HPLCanalysis. This can include the addition of a fusion tag to the proteinof interest to aid purification.

Useful purification methods have been exemplified here in the exampleswhere N-terminal hexa-histidine or N-terminal maltose binding protein(MBP) tags were used to facilitate purification using immobilized metalaffinity chromatography or amylose resin, respectively.

“A substantially pure protein” means a preparation which is at least 60%by weight (dry weight) the protein of interest. Preferably thepreparation is at least 75%, more preferably at least 80%, still morepreferably at least 90%, still more preferably at least 95%, mostpreferably at least 99% by weight of the protein of interest.

In some applications the protein product comprising the protein ofinterest may be used with the cell culture without recovery, isolationand/or without purification. In other applications the protein productcomprising the protein of interest may be recovered from the cellculture or cell medium or from host cells with or without purification.Furthermore, in some applications the protein product or purifiedprotein may be diluted or concentrated, or lyophilized.

This invention has multiple possible alternative solutions. In oneembodiment polycistronic vectors may be used i.e. vectors in which asingle mRNA encodes the protein of interest plus the sulfhydryl oxidaseand, where necessary, the thiol-disulfide isomerase (see FIG. 5). Theorder of the genes on polycistronic vectors does not affect the abilityto co-expression of the proteins.

In another embodiment, the sulfhydryl oxidase, and where necessary thethiol-disulfide isomerase, are on separate vector from the protein ofinterest (see FIG. 5). The use of a two vector system allows forseparate induction.

According to a preferred embodiment of the invention the vector orvectors is a plasmid or plasmids.

In one embodiment of the invention co-expression of a cytoplasmicallyexpressed sulfhydryl oxidase with the protein of interest in wild typeprokaryotic cells is undertaken. Disulfide bonds in the periplasm and inthe ER are thought to form sequentially i.e. cysteine 1 is linked tocysteine 2, cysteine 3 to cysteine 4 etc. Hence proteins with a smallnumber of sequential disulfide bonds should fold efficiently to theirnative state in the cytoplasm upon pre- or co-expression of acytoplasmically expressed sulfhydryl oxidase. This is exemplified herewith the efficient production of folded luminal domain of human tissuefactor and E. coli alkaline phosphatase upon co-expression of thesulfhydryl oxidase (see examples 2 and 3). The sulfhydryl oxidase may betargeted to the cytoplasm by the removal of the N-terminal signalsequence where necessary.

In another embodiment of the invention, co-expression of acytoplasmically expressed thiol-disulfide isomerase along with thesulfhydryl oxidase and the protein of interest is undertaken. Forproteins with an increased number of sequential disulfide bonds there isa greater probability that incorrect disulfide bonds will form. Theformation of incorrect disulfide bonds or the formation ofnon-sequential native disulfide bonds in the ER and periplasm is knownto require the subsequent action of a thiol-disulfide isomerase toattain the native disulfide state of the protein of interest.Co-expression of such an isomerase, for example DsbC or PDI targeted tothe cytoplasm, and combined with the expression of the sulfhydryloxidase and the protein of interest increases the yield of nativelyfolded protein. This has been exemplified with E. coli phytase (seeexample 4) and kringle t-PA (see example 6) and other proteins. Theisomerases may be targeted to the cytoplasm by the removal of theirN-terminal signal sequence.

Within the scope of the present invention are any enzymes withthiol-disulfide isomerase activity.

Preferred PDI sequences (Enzyme classification EC 5.3.4.1) comprisehuman PDI or yeast Pdi1p sequences. Yeast Pdi1p sequences can be foundfor example as Genbank accession numbers CAA42373 or BAA00723 orCAA38402.

Human PDI family members are for example PDI (for example as Genbankaccession numbers CAA28775 or AAC13652 (SEQ ID NO:11)), ERp57 (forexample as Genbank accession numbers BAA03759 or AAC51518), PDIp (forexample as Genbank accession numbers BAE38734 or AAK61223), ERp72 (forexample as Genbank accession numbers AAA58460 or AAH00425), PDILT (forexample as Genbank accession numbers BAC05068 or AAH42607), ERp27 (forexample as Genbank accession numbers AAQ88900 or AAH30218), PDIr (forexample as Genbank accession number BAA08451), ERp28 (for example asGenbank accession numbers CAA64397 or CAG46468), Erdj5 (for example asGenbank accession numbers AF038503 or AK027696), P5 (for example asGenbank accession numbers BAA08450 or AAH01312), ERp18 (for example asGenbank accession numbers AF543416 or CA117031), ERp44 (for example asGenbank accession numbers CAC87611 or AAQ89407), ERp46 (for example asGenbank accession numbers AAQ89009 or BAC11526), TMX (for example asGenbank accession numbers AAQ89003 or AK075395), TMX2 (for example asGenbank accession numbers AAD27740 or AAH00666), TMX3 (for example asGenbank accession numbers BAG53687 or AAH93792), TMX4 (for example asGenbank accession numbers AAQ89363 or BAC11599), hAG-2 (for example asGenbank accession numbers AAC77358 or AAQ89368) and hAG-3 (for exampleas Genbank accession numbers AAL55402 or AAH58284).

Sequences for DsbC (EC 5.3.4.1) can be found for example as Genbankaccession numbers AAA83074 or AAC75931 (SEQ ID NO: 10).

The thiol-disulfide isomerases having N-terminal signal sequence can beexpressed in the cytoplasm by the removal of the N-terminal signalsequence.

In addition to mature thiol-disulfide isomerases, shorter fragments ormodified forms or chimeric proteins may be used as long as they retainthiol-disulfide isomerase activity.

Enzymes having thiol-disulfide isomerase activity may comprise fragmentsor modifications of the above amino acid sequences.

“A modified protein” refers to a protein wherein at one or morepositions there have been amino acid insertions, deletions, orsubstitutions, either conservative or nonconservative, provided thatsuch changes result in a protein still having thiol-disulfide isomeraseactivity.

A modified version (or variant) of the enzyme having thiol-disulfideisomerase activity comprises typically an amino acid sequence having atleast 25%, preferably at least 40%, more preferably at least 50%, stillmore preferably at least 60%, still and still more preferably at least70%, more and more preferably at least 90% identity, most preferably atleast 95% or at least 98% identity to any of the above mentioned aminoacid sequences.

In addition to full length enzyme having thiol-disulfide isomeraseactivity, shorter fragments may be used as long as they retainthiol-disulfide isomerase activity. A fragment refers to a proteinhaving at one or more positions deletions. The fragment may comprise atleast 30%, at least 40%, at least 50%, preferably at least 60%, morepreferably at least 70%, still more preferably at least 80%, more andmore preferably at least 90%, or at least 95% of the complete amino acidsequence of the proteins.

According to other preferred embodiments of the invention, the enzymehaving thiol-disulfide isomerase activity of the invention is encoded bya nucleic acid sequence hybridizing under low or under high stringentconditions to nucleic acid sequences encoding the above mentioned aminoacid sequences. By high stringency conditions are meant conditions asdisclosed for example in Ausubel et al. Current Protocols in MolecularBiology, 1996, Wiley Sons, New York, N.Y. High stringency hybridizationconditions may comprise hybridization at about 42° C. and about 50%formamide, a first wash at about 65° C., about 2×SSC, and 1% SDS,followed by second wash at about 65° C., and about 0.1% SDS, 1×SSC.Lower stringency hybridization conditions may comprise hybridization atabout 42° C. in the absence of formamide, a first wash at about 42° C.,about 6×SSC, and 1% SDS, followed by second wash at about 50° C., about6×SSC and about 1% SDS.

Within the scope of the present invention are also nucleic acidsequences encoding the proteins comprising any of the amino acidsequences of the above gene bank accession numbers or the sequences SEQID NO:10 or SEQ ID NO:11 having thiol-disulfide isomerase activity, ornucleic acid sequences encoding a fragment of said sequences or amodified version of said sequences, which sequences still havethiol-disulfide isomerase activity. Suitable nucleic acid sequencesencoding proteins having thiol-disulfide isomerase activity are publiclyavailable and can be found in gene banks.

In another embodiment of the invention, the protein of interest isexpressed as a fusion protein. Disulfide bond formation is the ratelimiting step in protein folding in vitro and in vivo. If the foldingprotein misfolds and aggregates (forms inclusion bodies) before thesulfhydryl oxidase and/or thiol-disulfide isomerase can act on it thenlow yields of correctly folded protein will be obtained. The generationof a fusion protein between a well expressed and quickly folding proteinand the protein of interest is known to often inhibit inclusion bodyformation. Well known in the art examples include maltose bindingprotein (MBP), glutathione S-transferase (GST) and thioredoxin (Trx). Inthe examples included here we used MBP. The use of this fusion proteintechnology combined with co-expression of the sulfhydryl oxidaseincreased the yield of soluble protein for human CSF3 and BMP4 (seeexample 5). When this technology was combined with co-expression of athiol-disulfide isomerase as well it increased the soluble yields ofkringle t-PA (see example 6) and other proteins.

In another embodiment of the invention, mutant prokaryotic cells areused. Usually the cytoplasm of E. coli is reducing and any disulfidesthat are formed can be reduced by thioredoxin/thioredoxin reductaseand/or the glutathione/glutaredoxin/glutathione reductase systems whilethe protein is folding. To inhibit this, the expression of thesulfhydryl oxidase can be combined with knock-outs in these twopathways, for example the commercial rosetta-gami strain. Whenexpression of the sulfhydryl oxidase was combined with the rosetta-gamistrain background it increased the yields of active kringle tPA (seeexample 6) but decreased the yields of active alkaline phosphatase (seeexample 3) and phytase (see example 4).

In another embodiment of the system the sulfhydryl oxidase, and whereappropriate a thiol-disulfide isomerase, are expressed prior toexpression of the protein of interest. The kinetic partitioning betweenproductive folding and the formation of inclusion bodies is known todepend on the degree to which protein aggregates are already present inthe system. Upon co-expression of a sulfhydryl oxidase with the proteinof interest, the initial production of the protein of interest iseffective in the absence of sulfhydryl oxidase, since both are beingco-expressed. This increases the chances of initial aggregate formationwhich in turn becomes an auto-catalytic process. The presynthesis of thesulfhydryl oxidase, and thiol-disulfide isomerase if required, prior tothe induction of synthesis of the protein of interest circumvents thisproblem and would be expected to increase the solublebiologically-active yields of the protein of interest. Suitable vectorsbased on pLysS with an arabinose induction system to enable this wereconstructed (see FIG. 5). This system is able to fold a range ofproteins and increases the yield of active protein for example seekringle t-PA (example 6).

According to the present disclosure

-   -   The protein production system according to this disclosure        increases production of active protein in prokaryotic hosts        compared to wild type strains. The increase is over 100-fold in        a wild-type E. coli background. The present system also gives        circa 3 fold higher activity of protein of interest than the        prior system (rosetta-gami strain). This has been here        exemplified by expressing alkaline phosphatase, tPA and phytase        in this system and in the commercial rosetta-gami strain.    -   The rosetta-gami strain is genetically unstable and grows much        slower than the comparable wild-type strains. The use of wild        type strains modified according to the present disclosure is        more advantageous. Although, for some proteins the use of a        strain being deficient in thioredoxin reductase or glutathione        reductase activity and being modified according to the present        disclosure, may be of advantage.    -   In addition to sulfhydryl oxidase the expression of disulfide        isomerase may be of advantage, in particular if the protein of        interest comprises non-sequential disulfide bonds or a large        number of sequential disulfide bonds.    -   The more proteins that are required to be co-expressed, the        smaller the amount of cellular resources available to make the        protein of interest i.e. the system must always be kept to the        simplest possible.    -   The more disulfide bonds the target protein has the higher the        level of required expression of the sulfhydryl oxidase and the        disulfide isomerase. However, too high a level of expression of        either factor will result in a reduction in the levels of        expression of the target protein (see above).    -   Target proteins with non-optimal codon-usage for E. coli        production may require the co-expression of rare tRNA's for        optimal production e.g. the use of the pLysS RARE plasmid (found        in rosetta-gami along with the gor and trxB knockouts).    -   Target proteins that naturally have a pro-sequence generally may        require the use of a fusion protein to keep them soluble during        folding.

Various embodiments of the invention are described below with the aid ofthe following numbered clauses 1-19:

1. A method for producing a protein of interest containing one or moredisulfide bonds in its native state, which comprises that a prokaryotichost cell is genetically engineered to express the protein of interestand a sulfhydryl oxidase in the cytoplasm of the host cell, said proteinof interest being formed in a soluble form and containing disulfidebonds due to the presence of the sulfhydryl oxidase in the cytoplasm ofsaid host cell.2. The method according to clause 1, wherein the protein productcomprising the protein of interest is recovered from the cell culture orfrom the host cells and optionally purified.3. The method according to clause 1 or 2, wherein the protein ofinterest is co-expressed with the sulfhydryl oxidase.4. The method according to any one of clauses 1 to 3, wherein thesulfhydryl oxidase is expressed prior to the protein of interest.5. The method according to any one of the preceding clauses, wherein thehost cells are engineered to further express a thiol-disulfide isomerasein the cytoplasm.6. A prokaryotic host cell for producing a protein of interestcontaining natively folded disulfide bonds, which comprises that thehost cell is genetically engineered to express a sulfhydryl oxidase anda protein of interest in the cytoplasm of the host cell.7. The method or the host cell according to any one of the precedingclauses, wherein the sulfhydryl oxidase uses FAD as a cofactor andcontains one, or more, redox active disulfide bonds.8. The method or the host cell according to any one of the precedingclauses, wherein the sulfhydryl oxidase belongs to sulfhydryl oxidasescomprising ERV/ALR sulfhydryl oxidase domain.9. The method or the host cell according to any one of the precedingclauses, wherein the sulfhydryl oxidase belongs to ERO family ofsulfhydryl oxidases.10. The method or the host cell according to any one of the precedingclauses, wherein the host cell further expresses a thiol-disulfideisomerase in the cytoplasm.11. The method or the host cell according to clause 10, wherein thedisulfide isomerase is PDI, or DsbC.12. The method or the host cell according to any one of the precedingclauses, wherein the prokaryotic host is a gram negative bacterium, suchas E. coli.13. The method or the host cell according to any one of the precedingclauses, wherein the protein of interest is produced as a fusionprotein.14. The method or the host cell according to any one of the precedingclauses, wherein the sulfhydryl oxidase and the disulfide isomerase areinduced either separately or together with the protein of interest.15. The method or the host cell according to any one of the precedingclauses, wherein the host cell is deficient in thioredoxin reductaseand/or glutathione reductase activity.16. A vector system for a prokaryotic host cell for producing a proteinof interest containing natively folded disulfide bonds, which comprises

-   -   a vector encoding a protein of interest, or having a site for a        nucleic acid sequence encoding a protein of interest, and a        sulfhydryl oxidase, or    -   a first vector encoding a protein of interest, or having a site        for a nucleic acid sequence encoding a protein of interest, and    -   a second vector encoding a sulfhydryl oxidase,        wherein said vector or vectors are constructed to be capable of        expressing the protein of interest and the sulfhydryl oxidase in        the cytoplasm of a prokaryotic host cell.        17. The vector system according to clause 16 for producing a        protein of interest, wherein the system further comprises a        vector encoding a thiol-disulfide isomerase, said vector being        the same or different vector as the vectors in the vector        system, and said vector being constructed to be capable of        expressing a thiol-disulfide isomerase in the cytoplasm of the        prokaryotic host.        18. The vector system according to clause 16 or 17, wherein the        sulfhydryl oxidase and thiol-disulfide isomerase are induced        either separately or together with the protein of interest.        19. A prokaryotic host cell system, which comprises the vector        system according to any one of clauses 16 to 18.

The following non-limiting examples illustrate the invention.

EXAMPLES Example 1

Plasmid constructs used in the protein expressions were generated aspart of prestudies (see FIG. 5). The original constructs werepolycistronic with multiple genes encoded by a single mRNA driven by aT7 promoter system (IPTG inducible) in a modified version of pET23.These included the protein of interest alone (A), this plus thesulfhydryl oxidase (B) or plus the sulfhydryl oxidase and either PDI orDsbC as a thiol-disulfide isomerase (C).

Subsequent constructs had the sulfhydryl oxidase (D) or the sulfhydryloxidase plus the thiol-disulfide isomerase (E) on an arabinose promoteras part of a modified version of pLysS. Constructs D and E are fullycompatible with construct A and allow easy inter-conversion of theprotein of interest in the system. In addition, constructs D and E allowpre-expression or co-expression of the sulfhydryl oxidase, or thesulfhydryl oxidase and thiol-disulfide isomerase, and the protein ofinterest.

Example 2 Efficient Production of the Luminal Domain of Human TissueFactor

Tissue factor (TF), also known as thromboplastin factor III, is aprotein involved in the coagulation of blood. It is a transmembraneprotein whose luminal domain contains two sequential disulfide bonds.

pVD81, is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM)(SEQ ID NO: 11) followed in frame with the luminal domain of humantissue factor (sTF) as represented by the fragment Ser 33-Glu 251 of thefull length protein. This vector expresses sTF upon induction with IPTG.

pVD77 is a derivative of pVD81, in which the gene for the sulfhydryloxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) (SEQ IDNO: 5) after the gene for sTF (with suitable ribosome binding sites toinitiate translation of both; see FIG. 5). This polycistronic vectorco-expresses sTF and Erv1p upon induction with IPTG.

E. coli strains transformed with these expression vectors were streakedout from glycerol stocks stored at −70° C. onto agar plates containingsuitable antibiotics to allow for selection. The next day one colonyfrom these plates were used to inoculate 5 mls of LB media, containingsuitable antibiotics, and grown overnight at 30° C., 200 rpm. Thisovernight culture was used to seed a 50 mls culture of LB containingsuitable antibiotics in a 250 ml flask to an optical density of 0.05 at600 nm (OD₆₀₀). This culture was grown at 25° C., 200 rpm until theOD₆₀₀ reached 0.4 at which point protein production was induced by theaddition of 1 mM IPTG. The cells were then grown for 4 hours at 25° C.,200 rpm and the final OD₆₀₀ measured. The cells were collected bycentrifugation and resuspended to an OD₆₀₀ equivalent of 10 in 20 mMsodium phosphate pH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozyme,and frozen. Cells were lysed by freeze-thawing. Where appropriate, theinsoluble fraction was removed by centrifugation and the solublefraction removed quickly to a new container.

When sTF is expressed alone in the cytoplasm of the E. coli strain BL21(DE3) pLysS the protein is unable to fold correctly and forms insolubleinclusion bodies. When a sulfhydryl oxidase is co-expressed at 25° C.with sTF in the cytoplasm of the same E. coli strain, sTF is madesolubly in high yields. FIG. 6 shows representative SDS-PAGE fromexpression of sTF with co-expression with a sulfhydryl oxidase in LBmedia at 25° C. Lane 2 shows the total E. coli lysate, lane 1 shows thesoluble fraction. The position of the luminal domain of tissue factor ismarked with an arrow.

Purification of this protein via an N-terminal hexa-histidine tagfollowed by treatment with iodoacetamide and mass spectrometry analysisof the purified protein revealed the existence of no free thiol groupsimplying that two disulfide bonds had been formed in this system.

Example 3 Efficient Production of E. coli Alkaline Phosphatase

Alkaline phosphatase is a hydrolase which removes phosphate groups frommany types of molecules. The bacterial enzyme folds in the periplasm. Ithas two sequential disulfide bonds whose formation is essential foractivity. Since it is easily assayed bacterial alkaline phosphatase iswidely used as a model protein for disulfide bond formation in vivo.

pVD80, is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM)(SEQ ID NO:12) followed in frame with the mature form of E. colialkaline phosphatase (PhoA) as represented by the fragment Arg 22-Lys471 of the full length protein. This vector expresses alkalinephosphatase upon induction with IPTG.

pVD82 is a derivative of pVD80, in which the gene for the sulfhydryloxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) (SEQ IDNO: 5) after the gene for E. coli alkaline phosphatase (with suitableribosome binding sites to initiate translation of both; see FIG. 5).This polycistronic vector co-expresses E. coli alkaline phosphatase andErv1p upon induction with IPTG.

E. coli strains with these expression vectors were streaked out fromglycerol stocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point protein production was induced by theaddition of 1 mM IPTG. The cells were then grown for 4 hours at 30° C.,200 rpm and the final OD₆₀₀ measured. The cells were collected bycentrifugation and resuspended to an OD₆₀₀ equivalent of 10 in 20 mMsodium phosphate pH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozymeand frozen. Cells were lysed by freeze-thawing. Where appropriate, theinsoluble fraction was removed by centrifugation and the solublefraction removed quickly to a new container.

To monitor the production of active alkaline phosphatase we used thestandard 4-nitrophenylphosphate assay at pH 8.0. This assay revealedthat expression of alkaline phosphatase in the cytoplasm of E. coliresulted in the production of minimal active protein. In contrastco-expression of a sulfhydryl oxidase resulted in significant levels ofsoluble active protein (see FIG. 7).

FIG. 7 shows production of PhoA in the cytoplasm of E. coli in LB mediaat 30° C. Panel A) SDS-PAGE analysis. Alternate lanes show total (T) andsoluble (S) fractions. The final lanes show the purified protein fromBL21 with co-expression of Erv1p and the molecular weight markers. Thestrains used were BL=BL21 (DE3) pLysRARE and RG=rosetta-gami;+E=co-expression of the sulfhydryl oxidase Erv1p from a polycistronicvector. The positions of PhoA and Erv1p are marked with arrows. PanelB). Measurements for the activity of E. coli alkaline phosphataseexpressed in the cytoplasm of E. coli in LB media at 30° C.4-nitrophenylphosphate used as the substrate, pH of the reaction was 8.0and the values are cited as relative activity (%) compared with the mostactive system which is BL21 (DE3) pLysSRARE+expression of the sulfhydryloxidase. The strains used were BL=BL21 (DE3) pLysRARE andRG=rosetta-gami; +E=co-expression of the sulfhydryl oxidase Erv1p from apolycistronic vector. Panel C) Representative blot from a shift-assaybased on alkylation of free thiol groups to examine the disulfide bondstatus of the PhoA produced. The samples are treated with thethiol-blocking agent N-ethylmaleimide (NEM) before reduction andmaleimide based addition of polyethyleneglycol. Hence an increase inapparent molecular weight is consistent with the presence of one or moredisulfide bonds in the original sample. The greater the number ofdisulfide bonds the greater the mass shift. PhoA produced in both theΔgor ΔtrxB background and in the wild-type background plus co-expressionof Erv1p show a homogeneous disulfide bonded protein being produced,Panel D) Specific activity (μmole of product formed per minute per mg ofprotein) of PhoA purified from E. coli strains with and withoutco-expression of a sulfhydryl oxidase. The data is shown as mean±s.d.

Previously it had been shown that active alkaline phosphatase can bemade in strains such as rosetta-gami in which the reducing pathways havebeen disrupted. Production of alkaline phosphatase in rosetta-gamiresulted in the production of active protein, but circa two-fold lessactivity was seen than for the co-expression of the sulfhydryl oxidasein wild type E. coli.

Our system increases production of active protein over 100-fold in awild-type E. coli background and gives circa 2 fold higher alkalinephosphatase activity than the commercial rosetta-gami strain.

Example 4 Efficient Production of E. coli Phytase

Phytase (AppA) is an E. coli protein with similar activity to alkalinephosphatase except that it has optimal activity under acidic pH values(hence one of its alternative names is acid phosphatase). The proteincontains four disulfide bonds, one of which is non-sequential. As itcontains a non-sequential disulfide bond it is used as a model proteinfor disulfide bond formation which requires isomerisation. It is alsoused as an important animal feed adduct.

pVD96, is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM)(SEQ ID NO:12) followed in frame with the mature form of E. coli Phytase(AppA) as represented by the fragment Gln 23-Leu 432 of the full lengthprotein. This vector expresses phytase upon induction with IPTG.

pFH231 is a derivative of pVD96, in which the gene for the sulfhydryloxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) (SEQ IDNO: 5) after the gene for phytase (with suitable ribosome binding sitesto initiate translation of both; see FIG. 5). This polycistronic vectorco-expresses phytase and Erv1p upon induction with IPTG.

pFH244 is a derivative of pVD96, in which the gene for the mature formof DsbC from E. coli has been cloned (Asp 21-Lys 236) (SEQ ID NO: 10)after the gene for phytase (with suitable ribosome binding sites toinitiate translation of both; see FIG. 5). This polycistronic vectorco-expresses both phytase and mature DsbC upon induction with IPTG.

pFH233 is a derivative of pVD96, in which the gene for the sulfhydryloxidase Erv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO:5) and thegene for the mature form of DsbC from E. coli (Asp 21-Lys 236) (SEQ IDNO:10) have been cloned after the gene for phytase (with suitableribosome binding sites to initiate translation of both; see FIG. 5).This polycistronic vector co-expresses phytase, Erv1p and DsbC uponinduction with IPTG.

E. coli strains with these expression vectors were streaked out fromglycerol stocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point protein production was induced by theaddition of 1 mM IPTG. The cells were then grown for 4 hours at 30° C.,200 rpm and the final OD₆₀₀ measured. The cells were collected bycentrifugation and resuspended to an OD₆₀₀ equivalent of 10 in 20 mMsodium phosphate pH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozymeand frozen. Cells were lysed by freeze-thawing. Where appropriate, theinsoluble fraction was removed by centrifugation and the solublefraction removed quickly to a new container.

To monitor the activity of phytase an assay similar to that employed foralkaline phosphatase was used using 4-nitrophenylphosphate as thesubstrate (see example 3) except that the pH of the reaction was held atpH 2.5, the optimal for phytase activity.

FIG. 8 shows production of AppA in the cytoplasm of E. coli strains inLB media at 30° C. Panel A) Measurements for the activity of E. coliphytase (AppA). Absorbance values were measured after quenching thereaction with sodium hydroxide (NaOH) and the values are cites asrelative activity (%) compared with the most active system which is BL21(DE3) pLysSRARE+expression of the sulfhydryl oxidase and DsbC. Thebackground activity is subtracted. Strains used were BL=BL21 (DE3)pLysSRARE and RG=rosetta-gami. +=co-expression from a polycistronicvector where D=mature E. coli DsbC, E=S. cerevisiae Erv1p. B)Representative blot from a shift-assay based on alkylation of free thiolgroups to examine the disulfide bond status of the AppA produced. WhileAppA produced upon co-expression of Erv1p in a wild-type backgroundshows a homogeneous disulfide bonded protein being produced, the proteinproduced in the Δgor ΔtrxB background shows heterogeneity and a lowerdegree of disulfide bond formation. Note that the molecular weight ofthe mal-PEG is not homogenous and hence modified proteins, especiallythose with multiple mal-PEG added, appear as more defuse bands.Over-expression of phytase in the cytoplasm of E. coli results inactivity levels comparable with samples in which no phytase isexpressed. In contrast co-expression of a sulfhydryl oxidase results inincreased phytase activity (sample “BL+E”). Since phytase contains anon-sequential disulfide bond co-expression of a thiol-disulfideisomerase is required for optimal folding. Note however that in the wildtype background expression co-expression of only a thiol-disulfideisomerase does not significantly increase the activity of phytase(samples “BL+D” such proteins only aid so long as a sulfhydryl oxidaseis present (sample “BL+E+D” see FIG. 8).

The present invention increases production of active protein over100-fold in a wild-type E. coli background and gives more activity thanthe current commercial rosetta-gami system.

Example 5 Efficient Production of Soluble Human CSF3 and Human BMP4

Colony stimulating factors (CSF) are hormones that cause cells toproliferate and differentiate. CFS3 stimulates the production ofgranulocytes and stem cells and is also known as granulocytecolony-stimulating factor. It is used in oncological and hematologicalapplications and is made commercially under different names, includingNeupogen and Religast. CSF3 contains two sequential disulfide bonds andone free thiol group near the N-terminal of the protein which probablyresults in non-native disulfide bond formation.

Bone morphogenic proteins (BMP) are a group of cytokines which inducethe formation of bone and cartilage. BMP4 regulates the formation ofteeth, limbs and bones and plays a role in fracture repair. It containsthree non-sequential intra-molecular disulfide bonds along with oneinter-molecular disulfide bond.

pVD109 is a polycistronic derivative of pET23a which has a gene encodingfor a cytoplasmically targeted MBP tag (as represented by the fragmentLys 27-Thr 392 plus a linker encoding the amino acid sequenceNSSSNNNNHM, SEQ ID NO: 13) followed in frame with the mature form ofmature human CSF3 as represented by the fragment Ala 30-Pro 207 of thefull length protein. After this MBP-CSF3 fusion gene product the genefor the sulfhydryl oxidase Erv1p from S. cerevisiae has been cloned (Met1-Glu 189) (SEQ ID NO: 5), with suitable ribosome binding sites toinitiate translation of both; see FIG. 5. This polycistronic vectorco-expresses MBP-CSF3 and Erv1p upon induction with IPTG.

pVD110 is a derivative of pVD109, in which the gene for the mature formof DsbC from E. coli (Asp 21-Lys 236) (SEQ ID NO: 10) has been clonedbefore the gene for MBP-CSF3 (with suitable ribosome binding sites toinitiate translation of all; see FIG. 5). This polycistronic vectorco-expresses MBP-CSF3, Erv1p and DsbC upon induction with IPTG.

pVD111 is a derivative of pVD109, in which the gene for the mature formof PDI from H. sapiens (Asp 18-Leu 508) (SEQ ID NO: 11) has been clonedbefore the gene for MBP-CSF3 (with suitable ribosome binding sites toinitiate translation of all; see FIG. 5). This polycistronic vectorco-expresses MBP-CSF3, Erv1p and PDI upon induction with IPTG.

pVD112 is a polycistronic derivative of pET23a which has a gene encodingfor a cytoplasmically targeted MBP tag (as represented by the fragmentLys 27-Thr 392 plus a linker encoding the amino acid sequence NSSSNNNNHM(SEQ ID NO: 13) followed in frame with the mature form of mature humanBMP4 as represented by the fragment Pro 294-Arg 408 of the full lengthprotein. After this MBP-CSF3 fusion gene product the gene for thesulfhydryl oxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu189), with suitable ribosome binding sites to initiate translation ofboth; see FIG. 5. This polycistronic vector co-expresses MBP-BMP4 andErv1p upon induction with IPTG.

pVD114 is a derivative of pVD112, in which the gene for the mature formof DsbC from E. coli (Asp 21-Lys 236) (SEQ ID NO: 10) have been clonedbefore the gene for MBP-BMP4 (with suitable ribosome binding sites toinitiate translation of all; see FIG. 5). This polycistronic vectorco-expresses MBP-BMP4, Erv1p and DsbC upon induction with IPTG.

pVD113 is a derivative of pVD112, in which the gene for the mature formof PDI from H. sapiens (Asp 18-Leu 508) (SEQ ID NO: 11) have been clonedbefore the gene for MBP-BMP4 (with suitable ribosome binding sites toinitiate translation of all; see FIG. 5). This polycistronic vectorco-expresses MBP-BMP4, Erv1p and PDI upon induction with IPTG.

E. coli strains with these expression vectors were streaked out fromglycerol stocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point protein production was induced by theaddition of 1 mM IPTG. The cells were then grown for 4 hours at 30° C.,200 rpm and the final OD₆₀₀ measured. The cells were collected bycentrifugation and resuspended to an OD₆₀₀ equivalent of 10 in 20 mMsodium phosphate pH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozymeand frozen. Cells were lysed by freeze-thawing. Where appropriate, theinsoluble fraction was removed by centrifugation and the solublefraction removed quickly to a new container.

FIG. 9 shows SDS-PAGE from expression of the human CSF3 (lanes 2-7) andBMP4 (lanes 8-13) as fusion proteins with MBP in the cytoplasm of E.coli BL21 (DE3) pLysS with co-expression with a sulfhydryl oxidase in LBmedia at 30° C. and co-expression of PDI (lanes 4,5,10,11) or DsbC(lanes 6,7,12,13). Even numbered lanes show the total E. coli lysates,odd numbered lanes show the soluble fractions (except lane 1 which showsmolecular weight markers). The position of the MBP fusion proteins CSF3and BMP4 are marked with arrows. In E. coli BL21 (DE3) pLysS with noco-expression of a sulfhydryl oxidase no soluble protein expression canbe observed for either protein but our system allows soluble expressionof both.

The expression of CSF3 or BMP4 in the cytoplasm of E. coli BL21 (DE3)pLysS results in the formation of inclusion bodies. However, theexpression of either protein in fusion with MBP along with co-expressionof a sulfhydryl oxidase and the isomerase PDI or DsbC results inproduction of soluble protein (see FIG. 9).

Example 6 Efficient Production of Active Kringle tPA

Tissue plasminogen activator (tPA) is a protease that convertsplasminogen to plasmin, the major enzyme involved in the breakdown ofblood clots. It is used medically to treat pulmonary embolism,myocardial infarction and stroke. It is a large protein that contains 16non-sequential and one sequential disulfide bond and in addition has onefree thiol group in the native structure. It is available under thenames Activase and Retavase. Retavase is a fragment of tPA which onlycontains the kringle 2 and protease domains. As such it contains only 9disulfide bonds.

pKEHS1156, is a derivative of pET23a which has cloned into themulti-cloning site a gene encoding for an N-terminal hexa-histidine tag(MHHHHHHM) SEQ ID NO: 12 followed in frame with the form of Human tPAwhich contains the kringle 2 and protease domains as represented by thefragment Gly 211-Pro 562 of the full length protein. This vectorexpresses Kringle-tPA upon induction with IPTG.

pKEHS1165 is a derivative of pKEHS1156, in which the gene for thesulfhydryl oxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu189) after the gene for Kringle-tPA (with suitable ribosome bindingsites to initiate translation of both; see FIG. 5). This polycistronicvector co-expresses Kringle-tPA and Erv1p upon induction with IPTG.

pFH219 is a derivative of pKEHS1156, in which the gene for thesulfhydryl oxidase Erv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO:5) and the gene for the mature form of DsbC from E. coli (Asp 21-Lys236) (SEQ ID NO: 10) have been cloned after the gene for Kringle-tPA(with suitable ribosome binding sites to initiate translation of all;see FIG. 5). This polycistronic vector co-expresses Kringle-tPA, Erv1pand DsbC upon induction with IPTG.

pVD122 is a derivative of pET23a which has been cloned into themulti-cloning site a gene encoding for a cytoplasmically targetedN-terminal MBP tag (as represented by the fragment Lys 27-Thr 392 plus alinker encoding the amino acid sequence GSGSGSGSGSIEGRGSGSGSGSGSHM (SEQID NO: 14) followed in frame with the form of Human tPA which containsthe kringle 2 and protease domains as represented by the fragment Gly211-Pro 562 of the full length protein. This vector expresses theMBP-Kringle-tPA fusion protein upon induction with IPTG.

pVD163 is a derivative of pVD122, in which the gene for the sulfhydryloxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) afterthe gene for MBP-Kringle-tPA (with suitable ribosome binding sites toinitiate translation of both; see FIG. 5). This polycistronic vectorco-expresses MBP-Kringle-tPA and Erv1p upon induction with IPTG.

pVD171 is a derivative of pVD122, in which the gene for the mature formof DsbC from E. coli has been cloned (Asp 21-Lys 236) (SEQ ID NO: 10)after the gene for MBP-Kringle-tPA (with suitable ribosome binding sitesto initiate translation of both; see FIG. 5). This polycistronic vectorco-expresses MBP-Kringle-tPA and mature DsbC upon induction with IPTG.

pVD170 is a derivative of pVD122, in which the gene for the mature formof PDI from H. sapiens has been cloned (Asp 18-Leu 508) (SEQ ID NO: 11)after the gene for MBP-Kringle-tPA (with suitable ribosome binding sitesto initiate translation of both; see FIG. 5). This polycistronic vectorco-expresses MBP-Kringle-tPA and mature PDI upon induction with IPTG.

pVD164 is a derivative of pVD163, in which the gene for the mature formof DsbC from E. coli (Asp 21-Lys 236) (SEQ ID NO: 10) has been clonedafter the genes for MBP-Kringle-tPA and Erv1p (with suitable ribosomebinding sites to initiate translation of all; see FIG. 5). Thispolycistronic vector co-expresses MBP-Kringle-tPA, Erv1p and DsbC uponinduction with IPTG.

pVD165 is a derivative of pVD163, in which the gene for the mature formof PDI from H. sapiens (Asp 18-Leu 508) (SEQ ID NO: 11) have been clonedafter the genes for MBP-Kringle-tPA and Erv1p (with suitable ribosomebinding sites to initiate translation of all; see FIG. 5). Thispolycistronic vector co-expresses MBP-Kringle-tPA, Erv1p and PDI uponinduction with IPTG.

pFH256 is a derivative of pLysS in which the gene for sulfhydryl oxidaseErv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO: 5) and the gene forthe mature form of PDI from H. sapiens (Asp 18-Leu 508) (SEQ ID NO: 11)have been cloned under an arabinose inducible promoter/terminatorsystem. This vector allows pre-expression of Erv1p and PDI uponinduction with arabinose.

pFH255 is a derivative of pLysS in which the gene for sulfhydryl oxidaseErv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO: 5) and the gene forthe mature form of DsbC from E. coli (Asp 21-Lys 236) (SEQ ID NO: 10)have been cloned under an arabinose inducible promoter/terminatorsystem. This vector allows pre-expression of Erv1p and DsbC uponinduction with arabinose.

E. coli strains with these expression vectors (excluding strains whichcontain pFH255 or pFH256) were streaked out from glycerol stocks storedat −70° C. onto agar plates containing suitable antibiotics to allow forselection. The next day one colony from these plates were used toinoculate 5 mls of LB media, containing suitable antibiotics, and grownovernight at 30° C., 200 rpm. This overnight culture was used to seed a50 mls culture of LB containing suitable antibiotics in a 250 ml flaskto an optical density of 0.05 at 600 nm (OD₆₀₀). This culture was grownat 30° C., 200 rpm until the OD₆₀₀ reached 0.4 at which point proteinproduction was induced by the addition of 1 mM IPTG. The cells were thengrown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀ measured. Thecells were collected by centrifugation and resuspended to an (OD₆₀₀equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/ml DNase, 0.1mg/ml egg white lysozyme and frozen. Cells were lysed by freeze-thawing.Where appropriate, the insoluble fraction was removed by centrifugationand the soluble fraction removed quickly to a new container.

E. coli strains which contain pFH255 or pFH256 in addition to theexpression vectors for derivatives of tPA were streaked out fromglycerol stocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point pre-induction of Erv1p/PDI or Erv1p/PDI wasinduced by the addition of 0.5% arabinose. After 30 minutes productionof the tPA derivatives was induced by the addition of 1 mM IPTG. Thecells were then grown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀measured. The cells were collected by centrifugation and resuspended toan OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/mlDNase, 0.1 mg/ml egg white lysozyme and frozen. Cells were lysed byfreeze-thawing. Where appropriate, the insoluble fraction was removed bycentrifugation and the soluble fraction removed quickly to a newcontainer.

FIG. 10 shows analysis of human kringle tPA in various systems in LBmedia at 30° C. Activity measurements for the activity of human kringletPA expressed in the cytoplasm of E. coli using the Roche chromogenicsubstrate for tPA. The intrinsic protease activity of the E. coli lysatehas been subtracted. The human kringle tPA is expressed either as aprotein with an N-terminal his tag or as a protein with an N-terminalMBP-tag. The data is normalized to the equivalent of the best previouslyreported system, a ΔtrxB Δgor strain with co-expression of mature DsbC.When present the sulfhydryl oxidase Erv1p and/or mature DsbC areco-expressed from a polycistronic vector. Panel B). Panel A) SDS-PAGEfrom expression of the human kringle tPA as a fusion protein with MBP inthe cytoplasm of E. coli. BL21 pLysS RARE (BL) or rosetta-gami (RG),with and without co-expression with the sulfhydryl oxidase Erv1p andDsbC. Lanes marked with (T) and (S) show the total E. coli lysates andthe soluble fractions, respectively. The position of the MBP-kringle tPAfusion protein is marked with an arrow. There is a clear correlationbetween soluble expression of soluble fusion protein and activity.Panels C-E) Activity measurements for the activity of human kringle tPAexpressed in the cytoplasm of E. coli using the Roche chromogenicsubstrate for tPA. The human kringle tPA is expressed either as aprotein with an N-terminal his tag or as a protein with an N-terminalMBP-tag. The data is normalized to the equivalent of the best previouslyreported system, a ΔtrxB Δgor strain with co-expression of mature DsbC.Panel C) shows the effects of co- vs pre-expression of the sulfhydryloxidase Erv1p and mature DsbC. The strains used were BL21 pLysS RARE(BL) or rosetta-gami (RG) or origami (OG) with co-expression (+) orpre-expression (+p) for 30 minutes of the sulfhydryl oxidase Erv1p andmature DsbC. Note the increase in activity with pre-expression comesdespite the significant reduction in expression levels (see panel B) inorigami compared with rosetta-gami due to the expression of rare tRNAsin the later. Panel D) Relative activity of human kringle tPA producedin EnBase media (Biosilta Oy) at 30° C. Panel E) Relative yields ofactive protein produced in LB and EnBase media.

Expression of tPA or kringle tPA (similar but not identical to Retavase)in the cytoplasm of E. coli resulted in the production of insolubleinclusion bodies. Co-expression of a sulfhydryl oxidase increased theactivity of kringle tPA significantly (see FIG. 10). Combining this withco-expression of an isomerase, DsbC and/or with expressing kringle tPAas a fusion protein with Maltose binding protein (MBP) or using therosetta-gami or origami strains further increased the level of activeprotein produced (see FIG. 10) and the amount of soluble proteinproduced (see FIG. 10).

Example 7 Efficient Production of Soluble Folded BPTI and DisulfideMutants Therein

Bovine pancreatic trypsin inhibitor (BPTI) is a low molecular weightinhibitor of trypsin, kallikrein, chymotrypsin and plasmin. It isavailable under the name Trasylol and is used to inhibit coagulationduring bypass surgery. It is a small protein which contains threenon-sequential disulfide bonds. The folding intermediates of BPTI are ofacademic interest to understand protein folding pathways and especiallythe roles of protein folding catalysts in these processes.

pHEE12, is a derivative of pET23a which has cloned into themulti-cloning site a gene encoding for an N-terminal hexa-histidine tag(MHHHHHHM) SEQ ID NO: 12, followed in frame with the mature form ofBovine pancreatic trypsin inhibitor (BPTI) as represented by thefragment Arg 36-Ala 93 of the full length protein. This vector expressesBPTI upon induction with IPTG.

pHEE8, is a derivative of pHEE12 in which Cysteine 49 and Cysteine 73have been mutated to Alanine. This vector expresses C49A/C73A BPTI uponinduction with IPTG.

pHEE10, is a derivative of pHEE12 in which Cysteine 65 and Cysteine 86have been mutated to Alanine. This vector expresses C65A/C86A BPTI uponinduction with IPTG.

pKEHS1208 is a derivative of pLysS in which the gene for sulfhydryloxidase Erv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO: 5) and thegene for the mature form of PDI from H. sapiens (Asp 18-Leu 508) (SEQ IDNO: 11) have been cloned under an arabinose induciblepromoter/terminator system. This vector allows pre-expression of Erv1pand PDI upon induction with arabinose.

pKEHS1209 is a derivative of pLysS in which the gene for sulfhydryloxidase Erv1p from S. cerevisiae (Met 1-Glu 189) (SEQ ID NO: 5) and thegene for the mature form of DsbC from E. coli (Asp 21-Lys 236) (SEQ IDNO: 10) have been cloned under an arabinose induciblepromoter/terminator system. This vector allows pre-expression of Erv1pand DsbC upon induction with arabinose.

E. coli strains with these expression vectors (excluding strains whichcontain pKEHS1208 or pKEHS1209) were streaked out from glycerol stocksstored at −70° C. onto agar plates containing suitable antibiotics toallow for selection. The next day one colony from these plates were usedto inoculate 5 mls of LB media, containing suitable antibiotics, andgrown overnight at 30° C., 200 rpm. This overnight culture was used toseed a 50 mls culture of LB containing suitable antibiotics in a 250 mlflask to an optical density of 0.05 at 600 nm (OD₆₀₀). This culture wasgrown at 30° C., 200 rpm until the OD₆₀₀ reached 0.4 at which pointprotein production was induced by the addition of 1 mM IPTG. The cellswere then grown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀measured. The cells were collected by centrifugation and resuspended toan OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/mlDNase, 0.1 mg/ml egg white lysozyme and frozen. Cells were lysed byfreeze-thawing. Where appropriate, the insoluble fraction was removed bycentrifugation and the soluble fraction removed quickly to a newcontainer.

E. coli strains which contain pKEHS1208 or pKEHS1209 in addition to theexpression vectors for derivatives of BPTI were streaked out fromglycerol stocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point pre-induction of Erv1p/PDI or Erv1p/DsbC wasinduced by the addition of 0.5% arabinose. After 30 minutes productionof the BPTI derivatives was induced by the addition of 1 mM IPTG. Thecells were then grown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀measured. The cells were collected by centrifugation and resuspended toan OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/mlDNase, 0.1 mg/ml egg white lysozyme and frozen. Cells were lysed byfreeze-thawing. Where appropriate, the insoluble fraction was removed bycentrifugation and the soluble fraction removed quickly to a newcontainer.

When N-terminally hexa-histidine tagged wild-type mature BPTI or theC49A/C73A or C65A/C86A mutants, which mimic two of the two disulfidebond containing intermediates, are expressed in the cytoplasm of E. colithey form inclusion bodies. In contrast, when they are co-expressed witha sulfhydryl oxidase and a thiol-disulfide isomerase soluble protein isformed (see FIG. 11). Purification of the C49A/C73A or C65A/C86A mutantsusing immobilized metal affinity chromatography and subsequent analysisby reverse phase HPLC indicated that the purified proteins werehomogenous, with the elution point being equivalent to the relevant twodisulfide containing wild-type folding intermediate (see FIG. 11) i.e.only a single disulfide bond containing species which contained thecorrect disulfides was obtained.

Example 8 Efficient Production of Soluble Interferon α2 and Interleukin17

Interferon α2 is an anti-viral protein produced by macrophages. Itcontains 2 non-sequential disulfide bonds. Interleukin 17 is a cytokinethat induces the production of proinfamatory cytokines. It contains 2non-sequential disulfide bonds.

pGZ10 is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM)(SEQ ID NO: 12) followed in frame with the mature form of humaninterferon α2 as represented by the fragment Cys 24-Glu 188 of the fulllength protein. This vector expresses interferon α2 upon induction withIPTG.

pGZ15 is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for a cytoplasmically targeted N-terminal MBP tag(as represented by the fragment Lys 27-Thr 392 plus a linker encodingthe amino acid sequence GSGSGSGSGSIEGRGSGSGSGSGSHM) (SEQ ID NO: 14)followed in frame with the mature form of human interferon α2 asrepresented by the fragment Cys 24-Glu 188 of the full length protein.This vector expresses the MBP-interferon α2 fusion protein uponinduction with IPTG.

pMHR5 is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for a cytoplasmically targeted N-terminal MBP tag(as represented by the fragment Lys 27-Thr 392 plus a linker encodingthe amino acid sequence GSGSGSGSGSIEGRGSGSGSGSGSHM) (SEQ ID NO: 14)followed in frame with the mature form of human interleukin 17 asrepresented by the fragment Gly 24-Ala 155 of the full length protein.This vector expresses the MBP-interleukin 17 fusion protein uponinduction with IPTG.

pFH255 is a derivative of pLysS in which the gene for sulfhydryl oxidaseErv1p from S. cerevisiae (Met 1-Glu 189) and the gene for the matureform of DsbC from E. coli (Asp 21-Lys 236) have been cloned under anarabinose inducible promoter/terminator system. This vector allowspre-expression of Erv1p and DsbC upon induction with arabinose.

E. coli strains which contain pFH255 in addition to the expressionvectors for derivatives of interferon α2 or interleukin 17 were streakedout from glycerol stocks stored at −70° C. onto agar plates containingsuitable antibiotics to allow for selection. The next day one colonyfrom these plates were used to inoculate 5 mls of LB media, containingsuitable antibiotics, and grown overnight at 30° C., 200 rpm. Thisovernight culture was used to seed a 50 mls culture of LB containingsuitable antibiotics in a 250 ml flask to an optical density of 0.05 at600 nm (OD₆₀₀). This culture was grown at 30° C., 200 rpm until theOD₆₀₀ reached 0.4 at which point pre-induction of Erv1p/PDI orErv1p/DsbC was induced by the addition of 0.5% arabinose. After 30minutes production of the BPTI derivatives was induced by the additionof 0.5 mM IPTG. The cells were then grown for 4 hours at 30° C., 200 rpmand the final OD₆₀₀ measured. The cells were collected by centrifugationand resuspended to an OD₆₀₀ equivalent of 10 in 20 mM sodium phosphatepH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozyme and frozen. Cellswere lysed by freeze-thawing. Where appropriate, the insoluble fractionwas removed by centrifugation and the soluble fraction removed quicklyto a new container.

FIG. 12 shows SDS-PAGE analysis of production of interferon α2 andinterleukin 17 in the cytoplasm of the E. coli strain origami in EnBasemedia at 30° C. Lanes show total (T) and soluble (S) fractions of the E.coli lysates or protein purified (P) using an NTA-spin column or amylosecolumn (P). The positions of the interferon α2 and interleukin 17 fusionproteins are marked with arrows.

When N-terminally hexa-histidine or MBP-tagged mature interferon α2 orMBPtagged interleukin 17 are expressed in the cytoplasm of E. coli theyform inclusion bodies. In contrast, when there is pre-expression of asulfhydryl oxidase and mature DsbC in the origami strain of E. colisoluble protein is obtained (see FIG. 12). All three can be purified tohomogeneity by appropriate affinity chromatography.

Example 9 Efficient Production of Soluble Disulfide Linked Resistin

Resistin is a hormone linked to suppression of glucose uptake intoadipose cells by insulin. The mature protein contains 5 non-sequentialdisulfide bonds and an inter-molecular disulfide which links tworesistin monomers to together to form a disulfide linked homodimer.

pGZ16, is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for a cytoplasmically targeted N-terminal MBP tag(as represented by the fragment Lys 27-Thr 392 plus a linker encodingthe amino acid sequence GSGSGSGSGSIEGRGSGSGSGSGSHM) (SEQ ID NO:14)followed in frame with the mature form of human resistin as representedby the fragment Lys 19-Pro 108 of the full length protein. This vectorexpresses the MBP-resistin fusion protein upon induction with IPTG. Thefusion protein can be cleaved in the linker region by factor Xa.

pFH255 is a derivative of pLysS in which the gene for sulfhydryl oxidaseErv1p from S. cerevisiae (Met 1-Glu 189) and the gene for the matureform of DsbC from E. coli (Asp 21-Lys 236) have been cloned under anarabinose inducible promoter/terminator system. This vector allowspre-expression of Erv1p and DsbC upon induction with arabinose.

E. coli strains with these expression vectors (excluding strains whichcontain pKEHS1208 or pKEHS1209) were streaked out from glycerol stocksstored at −70° C. onto agar plates containing suitable antibiotics toallow for selection. The next day one colony from these plates were usedto inoculate 5 mls of LB media, containing suitable antibiotics, andgrown overnight at 30° C., 200 rpm. This overnight culture was used toseed a 50 mls culture of LB containing suitable antibiotics in a 250 mlflask to an optical density of 0.05 at 600 nm (OD₆₀₀). This culture wasgrown at 30° C., 200 rpm until the OD₆₀₀ reached 0.4 at which pointprotein production was induced by the addition of 0.5 mM IPTG. The cellswere then grown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀measured. The cells were collected by centrifugation and resuspended toan OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/mlDNase, 0.1 mg/ml egg white lysozyme and frozen. Cells were lysed byfreeze-thawing. Where appropriate, the insoluble fraction was removed bycentrifugation and the soluble fraction removed quickly to a newcontainer.

E. coli strains which contain pFH255 in addition to the expressionvectors for derivatives of resistin were streaked out from glycerolstocks stored at −70° C. onto agar plates containing suitableantibiotics to allow for selection. The next day one colony from theseplates were used to inoculate 5 mls of LB media, containing suitableantibiotics, and grown overnight at 30° C., 200 rpm. This overnightculture was used to seed a 50 mls culture of LB containing suitableantibiotics in a 250 ml flask to an optical density of 0.05 at 600 nm(OD₆₀₀). This culture was grown at 30° C., 200 rpm until the OD₆₀₀reached 0.4 at which point pre-induction of Erv1p/PDI or Erv1p/DsbC wasinduced by the addition of 0.5% arabinose. After 30 minutes productionof the BPTI derivatives was induced by the addition of 0.5 mM IPTG. Thecells were then grown for 4 hours at 30° C., 200 rpm and the final OD₆₀₀measured. The cells were collected by centrifugation and resuspended toan OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH 7.4, 20 μg/mlDNase, 0.1 mg/ml egg white lysozyme and frozen. Cells were lysed byfreeze-thawing. Where appropriate, the insoluble fraction was removed bycentrifugation and the soluble fraction removed quickly to a newcontainer.

FIG. 13 shows SDS-PAGE analysis of production of resistin in thecytoplasm of the E. coli strains in LB media at 30° C. Panel A)Analysis, under non-reducing and reducing conditions, of MBP-resistinfusion protein purified using an amylose resin column, indicates thatthe production of the disulfide linked resistin dimer increase withpre-expression of the sulfhydryl oxidase Erv1p and mature DsbC in bothwild-type and ΔtrxB Δgor strains. The strains used were BL21 pLysS RARE(BL) or origami (OG) with or without or pre-expression (+p) for 30minutes of the sulfhydryl oxidase Erv1p and mature DsbC. Panel B)Analysis, under non-reducing and reducing conditions, of MBP-resistinfusion protein purified using an amylose resin column of proteinproduced in origami with pre-expression of the sulfhydryl oxidase Erv1pand mature DsbC. The lanes show soluble (S) fractions of the E. colilysate, protein purified (P) using an amylose resin column and purifiedmaterial that has been digested with Factor Xa (D) to release MBP andresistin. Resistin is marked with an * in the digested fraction lanes.The position of the MBP-resistin fusion is indicated with an arrow.

Our invention allows efficient production of disulfide linked homodimersof resistin.

Example 10 Efficient Production of ¹⁵N Labeled Human Growth Hormone 1and Interleukin 6

Human growth hormone 1 (also known as somatotropin) plays a key role ingrowth control. It contains 2 sequential disulfide bonds. Interleukin 6is a cytokine with a wide variety of biological responses includingdifferentiation of B-cells, lymphocytes and monocytes. It contains 2sequential disulfide bonds.

pHIA487, is a derivative of pET23a which has cloned into themulti-cloning site a gene encoding for an N-terminal hexa-histidine tag(MHHHHHHM) (SEQ ID NO: 12) followed in frame with the mature form ofhuman growth hormone 1 as represented by the fragment Phe 27-Phe 217 ofthe full length protein. After this gene the gene for the sulfhydryloxidase Erv1p from S. cerevisiae has been cloned (Met 1-Glu 189) whichhas been codon optimized for expression in E. coli (with suitableribosome binding sites to initiate translation of both genes; see FIG.5). This polycistronic vector co-expresses hexahistidine tagged humangrowth hormone and codon optimized Erv1p upon induction with IPTG.

pYOE8 is a derivative of pET23a which has cloned into the multi-cloningsite a gene encoding for an N-terminal hexa-histidine tag (MHHHHHHM)(SEQ ID NO:12) followed in frame with the mature form of interleukin 6as represented by the fragment Val30-Met212 of the full length protein.After this gene the gene for the sulfhydryl oxidase Erv1p from S.cerevisiae has been cloned (Met 1-Glu 189) which has been codonoptimized for expression in E. coli (with suitable ribosome bindingsites to initiate translation of both genes; see FIG. 5). Thispolycistronic vector co-expresses hexahistdine tagged interleukin 6 andcodon optimized Erv1p upon induction with IPTG.

BL21 (DE3) pLysS E. coli strains with these co-expression vectors werestreaked out from glycerol stocks stored at −70° C. onto agar platescontaining suitable antibiotics to allow for selection. The next day onecolony from these plates were used to inoculate 20 mls of ¹⁵N-labelledM9 minimal media, containing suitable antibiotics, and grown overnightat 30° C., 200 rpm. This overnight culture was used to seed a 400 mlsculture of ¹⁵N-labelled M9 minimal media containing suitable antibioticsin a 2 L flask to an optical density of 0.1 at 600 nm (OD₆₀₀). Thisculture was grown at 30° C., 200 rpm until the OD₆₀₀ reached 0.4 atwhich point protein production was induced by the addition of 0.5 mMIPTG. The cells were then grown for 4 hours at 30° C., 200 rpm and thefinal OD₆₀₀ measured. The cells were collected by centrifugation andresuspended to an OD₆₀₀ equivalent of 10 in 20 mM sodium phosphate pH7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozyme and frozen. Cells werelysed by freeze-thawing. Where appropriate, the insoluble fraction wasremoved by centrifugation and the soluble fraction removed quickly to anew container.

Purification of ¹⁵N-labelled human growth hormone 1 and interleukin 6using immobilized metal affinity chromatography and subsequent analysisby reverse phase HPLC indicated that the purified proteins werehomogenous (see FIG. 14; upper panel interleukin 6, lower panel humangrowth hormone 1) i.e. only a single disulfide bond containing specieswas obtained. Parallel analysis by mass spectrometry and an Elman'sassay for free thiol groups under native and denaturing conditionsshowed that >98% of ¹⁵N-labelled was achieved for both proteins and thatthere were no detectable free thiols i.e. that two disulfide bonds wereformed in both proteins.

Our invention allows efficient production of disulfide containing humangrowth hormone and interleukin 6 from cytoplasmic expression in¹⁵N-labelled minimal media. Parallel expression showed similarproduction of disulfide bonded proteins in LB and EnBase media.

The invention claimed is:
 1. A method for producing a eukaryoticrecombinant protein of interest containing one or more disulfide bondsin a prokaryotic host, said method comprising expressing in thecytoplasm of the prokaryotic host cell a nucleotide sequence encodingthe eukaryotic protein of interest; and a nucleotide sequence encodingsequence of sulfhydryl oxidase catalyzing reactiondithiol+O₂→2disulfides+2H₂O; whereby said eukaryotic recombinant proteinof interest is formed in a soluble form and containing natively foldeddisulfide bonds in the cytoplasm of said host cell.
 2. The methodaccording to claim 1, wherein a protein product comprising the proteinof interest is recovered from a cell culture or from the host cells andoptionally purified.
 3. The method according to claim 1, wherein thesulfhydryl oxidase is co-expressed or expressed prior to the protein ofinterest.
 4. The method according to claim 1, wherein the sulfhydryloxidase uses FAD as a cofactor and contains one or more redox activedisulfide bonds.
 5. The method according to claim 1, wherein thesulfhydryl oxidase belongs to sulfhydryl oxidases comprising ERV/ALRsulfhydryl oxidase domain.
 6. The method according to claim 1, whereinthe sulfhydryl oxidase belongs to ERO family of sulfhydryl oxidases. 7.The method according to claim 1, wherein the host cell further expressesa thiol-disulfide isomerase in the cytoplasm.
 8. The method according toclaim 7, wherein the disulfide isomerase is eukaryotic ER-residentprotein disulfide isomerase PDI, or Thiol:disulfide interchange proteinDsbC.
 9. The method according to claim 1, wherein the prokaryotic hostis a gram negative bacterium.
 10. The method according to claim 1,wherein the protein of interest is produced as a fusion protein.
 11. Themethod according to claim 7, wherein expression of the sulfhydryloxidase and the disulfide isomerase is induced separately or togetherwith the protein of interest.
 12. The method according to claim 1,wherein the host cell is deficient of thioredoxin reductase orglutathione reductase activity or of both.
 13. The method according toclaim 9, wherein the bacterium is E. coli.